HARAMAYA UNIVERSITY

HARAMAYA INSTITUTE OF TECHNOLOGY

DEPARTMENT OF CHEMICAL ENGINEERING Project

Title:- Transformation of lignocellulosic biomass from food processing
activities into bioethanol

Prepared by

Name Id no.

1, Mekonnen Girma……………….….2340/12

2, Messay Deribew…………………….2404/12

3, Mitiku Zewdu……………………….2457/12

4, Muhammed Fentaw………………...2500/12

5, Mulualem Abebe………………...….2513/12

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 Declaration
We, the undersigned, hereby declare our commitment to the transformation of lignocellulosic
biomass from food processing activities into bioethanol through the implementation of a well-
defined project. Recognizing the growing importance of sustainable energy sources and the need
to reduce our dependence on fossil fuels, we aim to harness the untapped potential of agricultural
waste and residues generated from food production processes. By converting lignocellulosic
biomass into bioethanol, we strive to contribute to a greener and more sustainable future.

We are committed to ensuring that this project adheres to the highest environmental standards,
ensuring the conservation of natural resources, and mitigating potential environmental impacts.
Additionally, we will strive to engage with local communities, educating them about the benefits
of sustainable bioenergy production and addressing any concerns they may have.

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 Acknowledgement
First and foremost, we would like to thank our instructor Dr. M. Jayakumar, for providing us
with a helpful title that will aid us in writing our thesis.

Second we would like to express our sincere appreciation to all the individuals who have been
involved in this project in any capacity. Your guidance, feedback, and support have been
invaluable.

Last but not least we would also like to thank our friends. Without generous helpful and co-
operation our project would not have been a success.

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 Abstract
The transformation of lignocellulosic biomass from food processing activities into bioethanol has
gained increasing attention as a sustainable solution for energy production. The objective of this
project is to investigate the suitability of lignocellulosic biomass from food processing activities
as a feedstock for bioethanol production.

The extracted sugars are then fermented using specific yeast strains to produce bioethanol. The
efficiency of the fermentation process is assessed by analyzing key parameters like ethanol yield,
conversion rate, and overall process energy efficiency.

The implications of this project are significant in terms of waste management and renewable
energy production. Utilizing lignocellulosic biomass from food processing activities for
bioethanol production reduces the disposal burden on landfills, promoting a more sustainable
waste management system. Additionally, bioethanol derived from food waste can contribute to
reducing greenhouse gas emissions and dependence on fossil fuels.

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Table content

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List of table
Table 1: Different lignocellulosic biomass compositions (% dry basis)……………..…

Table 2: Comparison between the two main fermentation techniques………………….

List of figures
Fig.1. General composition of lingocellulosic biomass feedstock……………………..

Fig.2. Schematic pretreatment of lignocellulosic material…………………………….

Fig.3. Flowchart of cellulosic ethanol production from lignocellulosic biomass……..

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Abbreviations and symbols
AFEX Ammonia fiber explosion

Ca(OH)2 Calcium hydroxide

CO2 Carbon dioxide

CBH Cello bio hydrolase

CBP Consolidated biprocessing

CRI Cellulose crystallinity index

DMC Direct microbial conversion

DP Degree of polymerization

H2SO4 Sulferic acid

HMF Hydroxy-methyll furfural

IL Ionic liqued

NaOH Sodium hydroxide

OPF Oil palm frond

RO Severity factor

SO2 Sulfer dioxide

SHF Separet hyrolysis and fermentation

SSF Simultanous saccharafication and fermentation

SSCF Simultanous saccharafication co-fermentation

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 Chapter One
1. Introduction
1.1 Background
The need for alternative energy supplies has increased due to recent economic developments in
many countries worldwide. These trends are a result of the well-documented downsides of fossil
fuels, including their finite supply, greenhouse gas emissions and global warming, rising prices,
and unpredictable volatility. All of these drawbacks have increased interest in bioethanol and
other alternative fuels that are affordable, sustainable, and renewable. Bioethanol has a higher
octane number and heat of vaporization than gasoline, and it can be used as a fuel additive or in
conjunction with gasoline in engines designed for that purpose. Since ethanol is already mixed
with gasoline, car manufacturers have supported the development of vehicles that can run on a
mixture of up to 85% ethanol and 15% gasoline. As an oxygenated fuel, bioethanol may actually
be added to gasoline to raise its oxygen content, which improves hydrocarbon oxidation and
reduces greenhouse gas emissions. The second generation of bioethanol production uses cheap,
abundant, and renewable lignocellulosic materials as feedstock, as opposed to the first
generation's expensive starch and sugar generated from sugar cane and maize. Furthermore, the
human food supply chain is not adversely impacted by lignocellulosic materials because they do
not replace food in favor of producing bioethanol. Lignocelluloses have a complex structure
made up of cellulose, hemicelluloses, and lignin that resist breaking down. One of the best
strategies to convert such biomass into sugars is enzymatic saccharification due to its low energy
requirement and less pollution caused; but, the major problem is the low accessibility of

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cellulose because of rigid association of cellulose with lignin. This leads to difficulties within the
conversion process; therefore, breaking down lignin seal in order to make cellulose more
accessible to enzymatic hydrolysis for conversion is one main aim of pretreatment. In other
words, pretreatment is the crucial and costly unit process in converting lignocellulosic materials
into fuels. A suitable pretreatment procedure involves (1) disrupting hydrogen bonds in
crystalline cellulose, (2) breaking down cross-linked matrix of hemicelluloses and lignin, and
finally, (3) raising the porosity and surface area of cellulose for subsequent enzymatic
hydrolysis. Pretreatment techniques include chemical (alkali, acid, organosolv, ozonolysis, and
ionic liquid), physical (grinding and milling, microwave, and extrusion), physicochemical (steam
explosion, liquid hot water, ammonia fiber explosion, wet oxidation, and CO2 explosion), and
biological. However, a few inhibitory chemicals are created throughout the process, irrespective
of the pretreatment technique employed, and they have an adverse effect on the microbial
activity in the hydrolysis stage. Three main categories of inhibitors are identified: (1) weak acids,
including levulinic, acetic, and formic acids; (2) furan derivatives, including furfural and HMF
(5-hydroxy-2-methyll furfural); and (3) phenolic chemicals. This paper aims to present and
highlight some combination pretreatment approaches while reviewing several pretreatment
techniques for bioethanol production and providing a thorough analysis of the advantages and
disadvantages of each. Furthermore, it has been mentioned how important genetic and metabolic
engineering are to streamlining the pretreatment and hydrolysis processes, which leads to the
cost-effective synthesis of ethanol from lignocellulosic wastes.

1.2 Statement of problem

The transformation of lignocellulosic biomass from food processing activities into bioethanol
faces several challenges that need to be addressed for successful implementation. These
challenges include the inefficient conversion of biomass into bioethanol, high production costs,
lack of scalable and cost-effective technologies, and limited knowledge about the utilization of
specific biomass types. Additionally, the variability in the composition of lignocellulosic
biomass from different food processing activities poses a further obstacle to efficient bioethanol
production.

1.3 Objective

1.3.1 General objective:

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To transform lignocellulosic biomass from food processing activities into bioethanol

1.3.2 Specific Objectives:

1. To identify and evaluate different lignocellulosic biomass sources from food processing
activities that can be used for bioethanol production.

2. To optimize the pretreatment and enzymatic hydrolysis processes to maximize the yield of
fermentable sugars from lignocellulosic biomass.

3. To develop and test different fermentation strategies to convert the fermentable sugars into
bioethanol with high efficiency.

4. To assess the environmental and economic sustainability of the bioethanol production process
and compare it with other biofuel production methods.

1.4 Significance of the project

The transformation of lignocellulosic biomass from food processing activities into bioethanol
project is significant because it can contribute to reducing greenhouse gas emissions and
promoting renewable energy sources. It also offers a sustainable and efficient solution for the
management of food processing waste, which can be a significant environmental and economic
burden for industries. Additionally, this project can create opportunities for partnerships between
food processing industries and bioethanol production plants, leading to a more circular economy
and a more sustainable food system.

1.5 Lignocellulosic material composition

Cellulose
Cellulose (C6H10O5)x, the main constituent of lignocellulosic biomass, is a polysaccharide that
consists of a linear chain of D-glucose linked by β-(1,4)-glycosidic bonds to each other. The
Cellulose strains are associated together to make cellulose fibrils.

Cellulose fibers are linked by a number of intra- and intermolecular hydrogen bonds. Therefore,
cellulose is insoluble in water and most organic solvents.

Hemicelluloses
Hemicelluloses (C5H8O4)m, located in secondary cell walls, are heterogeneous branched
biopolymers containing pentoses (β-D-xylose, α-L-arabinose), hexoses (β-D-mannose, β-D-

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glucose, α-D galactose) and/or urgonic acids (α-D-glucuronic, α-D-4-O-methylgalacturonic and
a-D-galacturonic acids) . They are relatively easy to hydrolyze because of their amorphous, and
branched structure (with short lateral chain) as well as their lower molecular weight. In order to
increase the digestibility of cellulose, large amounts of hemicelluloses must be removed as they
cover cellulose fibrils limiting their availability for the enzymatic hydrolysis. Hemicelluloses are
relatively sensitive to operation condition, therefore, parameters such as temperature and
retention time must be controlled to avoid the formation of unwanted products such as furfurals
and hydroxymthyl furfurals which later inhibit the fermentation process.

Lignin
Lignin [C9H10O3(OCH3)0.9–1.7]n is an aromatic polymer synthesized from phenylpropanoid
precursors. The major chemical phenylpropane units of lignin consisting primarily of syringyl,
guaiacyl and p-hydroxy phenol are linked together by a set of linkages to make a complicated
matrix.

ENERGY 15-20%

35-50% Ash & other

Cellulose 15-20%
Lignin

20-35%

Hemicelluloses

Fig.1. General composition of lingocellulosic biomass feedstock.

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Fig.2. Schematic pretreatment of lignocellulosic material.

1.6 Influence of lignocellulosic biomass composition and structure on cellulose

hydrolysis and bioconversion
As mentioned earlier, lignocellulosic biomass composition plays a very crucial role in the
performance and efficiency of both pretreatment and biodegradation stages. Table 1 presents the
compositions of several suitable lignocellulosic biomass for bioethanol production. Utilization of
cellulose in native form, not only consumes large amount of enzyme but also results in low
cellulose enzymatic digestibility yield. Therefore, some structural modification of lignocellulosic
material (pretreatment), is required and to select a suitable pretreatment technology, recognition
of the main structural limiting factors is a critical step.

These factors include:

(1) specific surface area,

(2) cellulose crystallinity index (CrI),

(3) degree of polymerization (Dp),

(4) cellulose sheathing by hemicelluloses,

(5) lignin content and

(6) acetyl content.

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Table 1 Different lignocellulosic biomass compositions (% dry basis).

Lignocellulosic Cellulose Hemicellulose lignin

biomass glucan Xylan Arabinan Galacta Mannan Acid-insoluble Acid- soluble

Barley hull 33.6 30.5 6.1 0.6 Trace 19.3 ND

Barley straw 33.8 21.9 13.8

Corn cobs 33.7 31.9 6.1

Corn stover 38.3 21.0 2.7 2.1 ND 17.4

Cotton stalks 14.4 14.4 21.5

Wheat straw 30.2 18.7 2.8 0.8 ND 17

Rice straw 31.1 18.7 3.6 ND ND 13.3

Rye straw 30.9 21.5 ND ND ND 22.1 3.2

Oat straw 39.4 27.1 17.5

Soya stalks 34.5 24.8 9.8

Sunflower stalks 42.1 29.7 13.4

Switchgrass 39.5 20.3 2.1 2.6 ND 17.8 4.0

Sugarcane bagasse 43.1 31.1 11.4

Sweet sorghum bagasse 27.3 13.1 1.4 ND ND 14.3

Forage sorghum 35.6 18.4 1.8 ND ND 18.2

Olive tree pruning 25.0 11.1 2.4 1.5 0.8 16.2 2.2

Poplar 43.8 14.8 ND ND ND 29.1

Spruce 43.8 6.3 ND ND 14.5 28.3 0.53

Oak 45.2 20.3 ND ND 4.2 21.0 3.3

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 Chapter Two
2. Literature review
The process of turning lignocellulosic biomass into ethanol involves the following primary steps:
cellulose and hemicellulose hydrolysis into fermentable reducing sugars, sugar fermentation into
ethanol, lignin residue separation, and lastly, ethanol recovery and purification to fulfill fuel
requirements. Bacteria or yeasts often carry out the fermentation process, whereas lignocellulosic
enzymes typically do the hydrolysis. Porosity, or the accessible surface area of the waste
materials, cellulose crystallinity, the degree of cellulose and hemicellulose polymerization, and
the degree of hemicellulose acetylation are the parameters that have been shown to influence the
hydrolysis of cellulose and hemicelluloses. Many lignocellulosic biomass types have been
subjected to pretreatment techniques, and depending on the fermentation raw material, these
studies have produced a range of outcomes. This is a result of the diverse physico-chemical
characteristics of different lignocellulosic materials. Effective pretreatment of lignocellulosic
biomass should target these goals: (a) maximize the enzymatic digestibility of the pretreated
material; (b) partially depolymerize cellulose and hemicellulose; (c) solubilize hemicelluloses
and/or lignin; (d) modify the structure of lignin; (e) maximize the accessible surface area and
decrystallize cellulose; (f) minimize the loss of sugars; and (g) minimize capital and operating
costs. In addition to preventing the need to reduce the size of biomass particles and limiting the
development of toxic components that impede the growth of fermentative microorganisms, a
successful pretreatment must also retain the pentose (hemicellulose) fractions.

For various lignocellulosic biomasses, this project has concentrated on a few of the most popular
and significant physical, physio-chemical, chemical, and biological pretreatment methods. By
removing lignin from the biomass, these methods hope to create reducing sugars from cellulose
and hemicellulose that may be utilized to make bioethanol or other high-value products. These
pretreatment methods' basic principles, workings, advantages, disadvantages, and maximum
yield of sugars generated have all been discussed. The purpose of this analysis is to look at the
most recent advancements in the pretreatment processes used to produce bioethanol from
different lignocellulosic biomass sources. It will also look at how pretreatment, hydrolysis, and
fermentation interact with one another.

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2.1 Parameters for effective pretreatment of lignocellulosic biomass
There are several key factors which affect the rate of biological degradation of lignocelluloses.
The accessible surface area for enzymatic attack may be related to cellulose crystallinity, lignin,
and hemicellulose content.

Cellulose crystallinity
Both crystalline and amorphous areas are present in cellulose microfibrils, and it has been
suggested that cellulose crystallinity has a significant role in influencing how quickly relatively
refined cellulosic substrates hydrolyze. The majority of cellulose, or around two thirds of the
total, is crystallized. In actuality, cellulase is not as successful in breaking down the less
accessible crystalline component of cellulose as it is in hydrolyzing the more accessible
amorphous region. Therefore, it is predicted that cellulose with a higher crystallinity will be
more resistant to enzymatic hydrolysis, and it is generally agreed upon that lignocelluloses will
be more digestible when their crystallinity is reduced. However, it is not the only factor in
effective enzymatic hydrolysis of lignocellulosic biomass due to the heterogeneous nature of
celluloses and the contribution of other components such as lignin and hemicelluloses.

Effect of accessible surface area

Project has demonstrated a strong association between the enzymatic digestibility of
lignocellulosic materials and their pore volumes, which represent the accessible surface area for
cellulase and hemicellulase. Since lignin is eliminated, this link mostly benefits enzymatic
hydrolysis. There are two kinds of surface area in lignocellulosic biomass: internal and exterior.
The internal surface area of cellulosic fibers is dependent on their capillary structure, but the
exterior surface area is related to the size and form of the particles.

Effect of lignin
The integrity, structural stiffness, and ability to inhibit swelling of lignocellulosic material are all
attributed to the presence of lignin. Lignin covers the cellulose and hemicellulose. Lignin
reduces the effectiveness of hydrolysis by preventing enzymes from accessing cellulose and
hemicelluloses. The most significant known component contributing to lignocellulosic materials'
recalcitrance is lignin. Consequently, the pace and degree of enzymatic hydrolysis can be
enhanced by effective delignification procedures.

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Effect of hemicellulose
The cellulose fibers are shielded from enzymatic hydrolysis by hemicellulose, a physical barrier.
It has been demonstrated that hemicellulose removal raises the substrate's mean pore size, which
raises both the accessibility and likelihood of cellulose hydrolysis. Since lignin and acetyl groups
are connected to the hemicellulose matrix and may prevent polysaccharide breakdown, the
degree of acetylation in the hemicellulose is another crucial component.

2.2 Pretreatment methods

2.2.1 Physical pretreatment

The goal of physical pretreatments including freezing, grinding, chipping, milling, and radiation
is to make lignocellulosic materials more surface area and smaller particle sizes. Additionally, it
causes the feedstock's degree of polymerization and decrystallization to decrease. Usually, a
combination of various pretreatments and physical pretreatments is utilized.

Milling
Typically, the initial stage of pretreatment is milling. Several forms of milling are employed in
the processes of producing bioethanol, including ball milling, two-roll milling, hammer milling,
colloid milling, and disk milling. The type of physical pretreatment used determines the ultimate
particle size attained; for instance, following chipping, milling, or grinding, the particle size
decreases to 10–30 mm and 0.2–2 mm, respectively. The primary disadvantage of milling is its
high energy consumption. In order to get around it, wet disk milling has been presented as a
mechanical preparation that uses less energy. On the other hand, ball milling produces better
yields of glucose and xylose following the enzymatic hydrolysis of some pretreatment biomass
than wet disk milling does. Hideno et al., for example, studied the enzymatic hydrolysis of rice
straw following wet disk and ball mill pretreatment. The yields of xylose and glucose were
reported to be 89.4% and 54.3%, and 78.5% and 41.5%, respectively.

The efficacy of ball milling and wet disk milling for the treatment of sugarcane bagasse was
assessed by Sant'Ana da Silva et al. According to their analysis, the maximum glucose and
xylose yields for bagasse wet milling were 49.3% and 36.7%, respectively, whereas the optimal
glucose and xylose hydrolysis yields for bagasse processed by ball milling were 78.7% and
72.1%, respectively.

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Extrusion
A thermo-physical preparation known as extrusion involves the mixing, heating, and shearing of
materials to change their physical and chemical properties. The benefits of this approach are said
to include high shear, quick mixing, short residence time, moderate barrel temperature, lack of
furfural and HMF production, no washing or conditioning, flexibility to process change, ease of
scale-up, and most important possibility of continuous operation. Additionally, extrusion
produces no wastewater, which eliminates the need for wastewater disposal fees and solid waste.
Yoo et al. used an extrusion pretreatment (350 rpm screw speed, 80 1C maximum barrel
temperature, and 40% wet basis in-barrel moisture) for soybean hulls, and following enzymatic
hydrolysis, they obtained 94.8% glucose conversion (glucose yield of 0.37 g/g biomass).

Microwave
In place of traditional heating, microwave irradiation may be used to modify the cellulose's
ultrastructure, break down or partially eliminate lignin and hemicelluloses, disturb the surface's
silicified wax, and ultimately increase the sugars' enzymatic sensitivity to reduction. In theory,
traditional heating relies on surface heat transfer; nevertheless, similar to microwave heating,
heat is produced directly through the interaction of a heated item with an applied electromagnetic
field. More precisely, dielectric polarization caused by molecule collision is the primary
mechanism by which microwave irradiation breaks down cellulosic materials. The advantages of
this method include, (1) short process time, (2) high uniformity and selectivity (3) and less
energy input than the conventional heating.

Freeze pretreatment
The freeze pretreatment is a revolutionary strategy that has been developed lately for the physical
pretreatment of biomass and has been demonstrated to greatly boost rice straw's enzyme
digestibility. Although this pretreatment is expensive and has only been used in a small number
of trials, its special qualities lower environmental impact, less hazardous chemical application,
and high efficacy have garnered a lot of interest.

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2.2.2 Chemical pretreatment

Acid pretreatment
Acid pretreatment, primarily using sulfuric acid, is a widely used chemical for lignocellulosic
biomass. It converts polysaccharides into monosaccharides, increasing cellulose accessibility.
However, challenges include toxicity, corrosive equipment, acid recovery, degradation of
monosaccharides, and production of fermentation inhibitors like furan-type inhibitors. These
inhibitors can also break down into undesirable compounds at high temperatures. Diluted acid is
more desirable from an industrial standpoint since it produces less fermentation inhibitors, and as
a result, many investigations have been carried out using this method. For example, rice straw
was processed with 1% (w/w) sulfuric acid and allowed to sit at temperature of 160 ℃ or 180 ℃
for a duration of 3-5 minutes. This was followed by enzymatic hydrolysis, yielding a maximum
sugar yield of 83%.

Pretreatment with sulfuric acid (1.2%) and peracetic acid (1%), respectively, produced a 94%
total sugar yield in a research on rape seed straw and coastal Bermuda grass. The mixture had a
solid-to-liquid ratio of 1.5:10 and comprised 1% sulfuric acid and 1% acetic acid. The outcomes
demonstrated cellulose degradation of less than 15% and effective hemicellulose removal of
more than 90%. A research comparing the hydrolysis yields of wheat straw treated with organic
acids such as fumaric and maleic acids to sulfuric acid showed that these acids might potentially
be employed.

Alkaline pretreatment
This pretreatment method removes lignin, acetyl groups, and uronic acid substitutions that hinder
cellulose accessibility for enzymatic saccharification. It reduces solubilization of hemicelluloses
and cellulose, and causes chemical swelling of fibrous cellulose. This leads to saponification and
salvation reactions, disrupting crosslinks and increasing biomass porosity. Alkaline pretreatment
operates at lower temperatures and requires less complex reactors, but has long residence times
and requires neutralization of the slurry. Common pretreatment materials include sodium
hydroxide, potassium hydroxide, calcium hydroxide, and ammonia. NaOH (4-40 g/100 g dry
straw) was used to pretreat soybean straw at room temperature, resulting in a 64.55% glucose
yield and a 46.37% xylan elimination. In a different investigation, a total reducing sugar yield of
71% was obtained by pretreating Costal Bermuda grass for 15 minutes with a 0.75% NaOH

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solution. Furthermore, xylan and glucan had total conversion efficiencies of 65.11% and 90.43%,
respectively. In another study, the combination of NaOH and CaOH 2 (0.10 and 0.02 g/g raw bio
mass, respectively) was utilized to increase the alkaline pretreatment of switchgrass under
ambient conditions' cost-effectiveness. After pretreating the biomass with NaOH for a first time
and then Ca(OH)2 for the regenerated biomass, the yields of glucose and xylose were 59.4% and
57.3%, respectively.

Ionic liquid (IL) pretreatment

The pretreatment of lignocellulosic bio material using ionic liquids (ILs) has garnered significant
interest lately due to its ability to remove both lignin and carbs concurrently. The organic salts
known as ILs (melting point 100 °C) are made up of both cations and anions, usually tiny
inorganic anions and big organic cations. Indeed, two main parameters that might impact the
physical, chemical, and biological aspects of ILs are the degree of anion charge delocalization
and the cation structure (the symmetry and length of alkyl substituents, the presence of
hydrophobic groups, etc.). Overall, the main variables influencing the interaction between the
lignocellulosic materials and the ILs utilized are temperature, time spent in the pretreatment
process, cations, and anions. By forming strong hydrogen bonds, ILs containing formate, acetate,
chloride, or alkylphosphonate inions might degrade cellulose. In another way, the complex
network of cellulose, hemicelluloses, and lignin is broken down as a result of hydrogen bonds
that occur between the hydroxyl protons of sugars and the non-hydrated ions of ILs. Due to their
exceptional ability to dissolve cellulose, among the ILs examined, 1-allyl-3-methylimidazolium
chloride ([AMIM]Cl), 1-ethyl-3-methylimidazolium acetate ([EMIM]Ac), 1-butyl-3-
methylimidazolium chloride ([BMIM]Cl), and 1-ethyl-3-methyl imidazolium diethyl phosphate
([EMIM]DEP) have recently attracted a lot of attention. Switchgrass pretreated with [EMIM]Ac
resulted in glucan contents ranging from 50.4% to 67.7% in one research. It was also discovered
that this IL could remove 69.2% of the total lignin in switchgrass after three hours at 160℃. Up
to 25 weight percent of cellulose, [BMIM]Cl has been described as one of the finest cellulose
solvents. In a different study on oil palm frond (OPF) pretreatment using [BMIM]Cl, cellulose
crystallinity reduction and improved enzymatic digestibility was accomplished. As for lignin, 1-
butyl-3- methylimidazolium trifluoromethanesulfonate and 1,3-dimethylimidazolium
methylsulphate have been reported as the best solvents.

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Ionic liquids (ILs) pretreatment causes a minor shift in the biomass structure, with lignin,
hemicellulose, and cellulose percentages in OPF changing before and after treatment. This is
because regenerated cellulose becomes porous and amorphous, which increases its vulnerability
to cellulase breakdown. Among the benefits of ILs include low vapor pressure, good thermal and
chemical stability, and less hazardous process conditions. They are readily recyclable, necessitate
modest operating conditions, and are non-derivatizing. Low viscosity reduces energy usage by
dissolving cellulose at low temperatures. Negative side effects, including decreased IL stability
and adverse responses, can be brought on by high temperatures. The industrial use of ILs is
confronted with obstacles such costly IL quantities, energy-intensive recycling, and viscous
solution during pretreatment.

Organosolv pretreatment
In the organosolv method, organic solvents such as methanol, ethanol, acetone, ethylene glycol,
and tetrahydrofurfuryl alcohol may be utilized with or without the inclusion of a catalytic agent.
Lime, ammonia, sodium hydroxide, and sulfuric and hydrochloric acids are examples of bases
and organic or inorganic acids that are utilized as catalysts. This pretreatment is particularly
effective for high lignin lignocellulosic biomass because it may dissolve the internal linkages
between hemicelluloses and lignin. Furthermore, lignin of a high caliber and relative purity can
be produced as a byproduct of this procedure. It goes without saying that the elimination of
lignin increases cellulose's surface area, which increases its enzyme accessibility. The low
boiling point of organic solvents, the significant danger of operating at high pressure, and the
flammability and volatility of these solvents are the key disadvantages of this approach.

Additionally, it is important to recycle solvents to save operating costs and avoid inhibitory
effects on microorganisms and enzymatic hydrolysis. In a separate study, Pinus radiata was
subjected to a 1:1 acetone to water molar ratio pretreatment at 195 °C and pH 2.0 for five
minutes, resulting in an ethanol yield of almost 99.5%.

Ozonolysis
Ozone gas is used as an oxidant in this pretreatment to break down lignin and hemicelluloses and
improve the biodegradability of cellulose. Ozone gas has several benefits, including being a
potent oxidant, soluble in water, and readily accessible. Additionally, it could degrade lignin and
liberate soluble, lower molecular weight substances like formic and acetic acid. Where

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enzymatic hydrolysis yields of up to 88.6% and 57% were obtained compared to 29% and 16%
in non-ozonated wheat and rye straw, respectively. Lignin degradation of 49% was observed
when maize stover was broken down by ozonolysis. The primary benefit of this approach is the
absence of degradation byproducts, which reduces complexity in the hydrolysis stages that
follow. Besides, this process is carried out at ambient condition. On the other hand, the main
disadvantage is the cost of ozone used as a large amount of ozone is employed to treat
lignocellulosic materials.

2.2.3 Physico-chemical pretreatment

Steam explosion pretreatment

This is a well-studied thermo-mechanochemical process that breaks down structural elements by
auto-hydrolysis (chemical) of glycosidic bonds, steam-heating (thermo), and shearing (mechano;
caused by abrupt decompression and moisture evaporation). More precisely, pressured steam
(20-50 bar, 160-270 °C) is used to heat biomass particles for a few seconds to several minutes.
After that, the pressure is released to ambient pressure, condensed moisture evaporates, and the
lignocellulosic matrix separates. Because of the high temperature during this pretreatment, it is
thought to produce hemicellulose hydrolysis, convert lignin, and increase cellulose's crystallinity
by encouraging the crystallization of its amorphous sections. The parameters affecting steam
explosion efficiency are particle size, temperature (T) and residence time (t) and the combined
effect of both temperature and residence time is described by severity factor (R 0).
t

(Log R0) R0 = ∫ exp [ (T −100 ) /14.75 ] dt

0

Organic acids, such as acetic acids produced from the hydrolysis of acetyl groups linked to the
hemicellulose and formic and levulinic acids formed from other functional groups, are
responsible for the hydrolysis of hemicellulose during steam explosion pretreatment. At high
temperatures, water exhibits certain acidic characteristics and functions as an acid as well.
Lastly, by exposing the cellulose surface, the removal of hemicellulose from the cellulose
microfibril surface enhances the accessibility of enzymes and the rate at which cellulose is
hydrolyzed by enzymes. However, the breakdown of carbohydrates into furfural and HMF may
also occur throughout the process because of the current acidic circumstances.

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In order to achieve high sugar yields from some lignocellulosic biomass, such as softwood, an
acid catalyst like H2SO4 or SO2 must be added before the steam explosion process. Softwood's
hemicellulose has low acetyl group content, leading to higher inhibitory compounds that affect
fermentation. To remove these inhibitors, lignocellulosic biomass needs to be washed with water
after pretreatment. A study found that steam explosion pretreatment at 200 ℃ for 5 minutes
enhanced cellulose conversion to glucose by nearly four times at 80%, achieving an ethanol yield
of about 90%. The optimal conditions for steam explosion pretreatment of wheat straw were
180℃ and 10 minutes, resulting in an overall sugar yield of 85%. Steam explosion is a cost-
effective pretreatment technology for pilot scale demonstration and commercialized applications.

Ammonia fiber explosion (AFEX) pretreatment

AFEX is a pretreatment technique for biomass that uses liquid ammonia and is based on the idea
of the steam explosion process. For a few minutes, there is high pressure and mild temperature,
and then there is an abrupt pressure release. The lignin carbohydrate complex is broken down by
the fast expansion of ammonia gas, which increases the digestibility of biomass fibers. Because
of its low boiling point, AFEX does not immediately release sugars, but instead generates solid
material. It has been demonstrated to provide greater conversion rates for different types of
cellulosic biomass but less effective conversion rates for biomass with a high lignin component.
Poplar and maize stover perform best under the following circumstances, according to studies:
2:1 ammonia to biomass loading, 233% moisture, 180 °C; and 1:1 ammonia to biomass loading,
60% moisture, 90 ℃ respectively.

CO2 explosion pretreatment

Super-critical CO2 explosion is similar to AFEX and steam explosion pretreatments due to CO 2
molecules similar size properties to water and ammonia. It requires lower temperatures and is
less costly than AFEX, making it an ideal choice. CO 2 explosion also has non-toxicity and non-
flammability advantages. It has a liquid-like density and gas-like transport properties. CO 2
pretreatment, while maintaining acid-catalyzed processes, forms carbonic acid, which is less
corrosive due to its specific properties. It also removes CO 2 easily, creating no waste products
and requiring no further recovery.

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Liquid hot water
Hot compressed water, commonly referred to as liquid hot water, is a stream pretreatment
technique. However, as its name suggests, water is utilized instead of steam at high temperatures
of 170-230 °C and pressures of up to 5 Mpa. Additionally, numerous listed as aqueous
fractionation, hydrothermal pretreatment, aquasolv, and hydrothermal thermolysis. It provides
more easily available cellulose and hemicellulose by removing lignin. One efficient way to treat
several biomass kinds, including softwoods, is with liquid hot water.

Materials that are insoluble in water, solids that have been enhanced with cellulose, liquid
fractions like water, inhibitors, and solubilized hemicellulose are during the pretreatment
process, formed as slurry. It is best to keep the pH of liquid hot water between 4 and 7 to prevent
the production of inhibitors and the breakdown of sugar.

When pretreating maize stover in hot water for 15 minutes at 190°C, and managed to get the
most solubilized hemicellulose with the least amount of inhibitor formation. Through the
enzymatic hydrolysis of cellulose, they were able to convert it to glucose at a rate of 90%.
Because this pretreatment procedure does not include the use of a chemical catalyst or acid, it is
both economically and environmentally advantageous). Its benefits include low-temperature
requirements, the absence of inhibitory chemicals at high temperatures, and inexpensive liquid
hot water process solvents.

Wet oxidation
In the wet oxidation pretreatment method, biomass is heated to 170-200 °C and then placed
under high pressure (500-2000 kPa) for 10-15 minutes. It's simple to use wet oxidation. Earlier,
this has been used for soil cleanup and treatment of wastewater. With this method, the
lignocellulosic material is broken down, producing fewer inhibitors, less lignin, and less
cellulose. As a result, it creates an environment that is ideal for processes like fermentation and
enzymatic hydrolysis to operate on the pretreated product. This approach does have several
typical drawbacks, though, including capital expense and energy efficiency. The lignin-enriched
biomass residue is appropriate for the wet oxidation process. Three parameters, namely
temperature, reaction time, and oxygen pressure, affect how effective this method is. When the
temperature rises over 170 °C, water acts like an acid and catalyzes hydrolytic processes.

16
Hemicellulose is broken down, resulting in the formation of tiny pentose monomers. Wet
oxidation pretreatment has a greater impact on the lignin than the cellulose.

In addition to these, other chemical agents used in wet oxidation, such as sodium carbonate and
alkaline peroxide, lower the reaction temperature, promote hemicellulose breakdown, and result
in less inhibitor formation, such as furfurals and furfurl aldehydes. The wet oxidation condition
for the manufacture of ethanol from rice husk has been optimized by Banerjee et al. (2009). After
using this pretreatment technique, sugar output reductions of more than 70% were observed.
Alkaline peroxide-assisted wet air pretreatment of rice husk resulted in lignin and hemicellulose
solubilization of 88 and 67%, respectively. Compared to rice husk without treatment, the glucose
quantity increased 13 times.

2.2.4 Biological pretreatment

The breakdown of lignocellulose biomass is accomplished by means of natural microorganisms
with enzymes (bacteria, brown-white, and soft rot fungus) that are able to deconstruct cell walls.
The biological pretreatment method produces no unwanted chemicals, in contrast to chemical
and physical pretreatment.

Furthermore, no high pressure, acids, alkalis, high temperatures, or other reactive species are
required for this preparation. The lignin-degrading enzymes found in the fungi that produce
white and soft rot include laccases, polyphenol oxidases, manganese-dependent peroxidases, and
lignin peroxidases. These enzymes are effective in breaking down lignin. The features, mode of
action, and rate of breakdown by microbes are all determined by the targeted biomass
component. For example, brown-rot fungi mainly attack cellulose, but white and soft-rot fungi
are believed to be more helpful in breaking down lignin because they use enzymes that break
down lignin.

When low cellulose degrading and high lignin degrading fungi were pretreated on wheat straw,
the concentration of inhibitors decreased and the amount of fermentable sugar increased,
respectively. Ethanol generation and volumetric productivity with Pichia stipitis were 0.48 g/g
and 0.54 g/L h, respectively. Since biological pretreatment doesn't need the use of chemicals, it is
more effective than other pretreatment techniques. Moderate environmental conditions and low
energy requirements are also seen positively. The main barrier to the development of biological
approaches, however, is the low hydrolysis rate seen in biological materials. Along with lignin,

17
most bacteria that degrade lignin also break down cellulose and hemicellulose. Large-scale
commercial lignocellulose biomass biological processing using these microorganisms
necessitates intensive investigation.

2.2.5 Combination of pretreatment methods

It seems that the intended result cannot be achieved with a single pretreatment method.
Combining several pretreatment methods is a common practice in the lignocellulose-based
bioethanol manufacturing process. The biomass must first be mechanically reduced in size by a
procedure like milling, cutting, and crushing before being immersed in an alkaline or acidic
solution. An explosion of steam may optionally occur after this procedure (alkaline pretreatment
is preferable). Next comes cleaning and neutralization of the pretreated material. Every time this
procedure is used, a puffed material ready for further fermentation is produced. The reusability
of internal waste was also addressed in order to reduce chemical costs and increase the
sustainability of lignocellulosic bioethanol production.

2.3 Producing bioethanol from processed lignocellulose

Pretreated biomass may be converted into bioethanol using two different processes: hydrolysis
and direct microbial conversion (DMC), in addition to fermentation. In actuality, the DMC
method is time-consuming, offers low conversion rates, and carries a significant risk of
contamination. On the other hand, enzymatic hydrolysis in conjunction with microbial
fermentation is a recommended strategy that has shown to be more effective.

Because lignocellulosic biomass contains a greater quantity of cellulose and hemicellulose

sugars than starch or sugar-based biomass, producing bioethanol from it is a financially feasible
process. The structure of the plant cell wall is complicated, which raised the cost of manufacture.
There are two steps involved in the bioconversion of lignocellulosic materials to bioethanol. To
separate the different components of the biomass, it is first treated.

Scarification of lignocellulose
The polysaccharide-enriched substance is broken down by enzymes into single sugars (hexoses
and pentoses) after being treated with lignocellulose. The commercial enzyme used to hydrolyze
cellulose and hemicellulose is called cellulase, a name used to describe a broad range of enzymes
generated from microorganisms. These enzymes hydrolyze carbohydrates by either inverting or

18
maintaining the glycosidic bonds; the latter mechanism necessitates the synthesis of a glycosyl-
enzyme intermediate and is often completed in two phases.

In contrast to hemicellulose, which hydrolyzes more readily, cellulose is fairly durable and has a
crystalline structure that makes it resistant to depolymerization. To efficiently hydrolyze
cellulose using enzymes, the following three cellulose enzymes are commonly combined:

 Exo-1,4-β-D-glucanases including 1,4-β-D-glucan glucohydrolase to free D-glucose from

1,4-β-D-glucan and slowly hydrolyze D-cellobiose, and enzyme 1,4-β-D-glucan
cellobiohydrolase (CBH) to free cellobiose from 1,4-β-glucan
 β-D-Glucosidase or β-D-glucoside glucohydrolase to form D-glucose from
 Endo-1,4-β-glucanases or 1,4-β-D-glucan 4-glucanohydrolases. This enzyme randomly
breaks the 1,4-β-glucan bonds.

Despite a little variation in the ideal temperature for the two processes, enzymatic hydrolysis
promotes the development of microorganisms during fermentation.

Fermentation
Biomass processing (pretreatment and hydrolysis) is an essential step to optimize the
fermentation process (Gamage et al. 2010 ). Lignocellulosic biofuel technology faces a
bottleneck due to the lack of potent microorganisms capable of fermenting both pentose and
hexose sugars. To commercialize this technology, ideal microorganisms must meet criteria such
as wide substrate utilization, high ethanol yield, ethanol tolerance, and cellulolytic activity.
Currently, C6 sugars are easily fermented into bioethanol, but bioconversion of C 5 sugars is a
recent approach. Novel microorganisms like Pichiastipitis, Candida shehatae, and
Pachysolantannophilus have been screened and characterized for their ability to ferment both
sugars. Thermophilic microorganisms are ideal candidates for industrial bioethanol production
due to their ability to tolerate high temperatures and toxic by-products.

Currently, the main techniques that are usually involved in the fermentation of biomass
hydrolysates are separate hydrolysis and fermentation (SHF) and simultaneous saccharification
and fermentation (SSF). SSF is considered as a superior, cost-effective, and efficient technique
for the production of bioethanol than SHF because it prevents the formation of inhibitory by-
products and does not involve the use of separate reactors. However, any fluctuation in optimum
temperature conditions of enzymes for hydrolysis and fermentation diminishes its efficiency
19
(Neves et al. 2007 ). Buaban et al. ( 2010 ) reported that ethanol yield coefficient is higher under
SSF than SHF because of more conversion of pentose sugar (xylose) into xylitol. Comparative
study among these two techniques (SHF and SSF) is presented in Table 2.

Table 2 Comparison between the two main fermentation techniques

Fermentation process Features and advantages Limitations

Simultaneous saccharification Low costs Higher ethanol yields Difference in optimum

and fermentation due to removal of end product temperature conditions of
inhibition of saccharification enzyme for hydrolysis and
step fermentation.

Separate hydrolysis and Reduces the number of reactors End product inhibition
fermentation required Each step can be minimizes the yield of ethanol.
processed at its optimal Chance of contamination due to
operating conditions Separate long period process
steps minimize interaction
between the steps

Other methods for turning biomass into bioethanol include simultaneous saccharification and co-
fermentation (SSCF) and consolidated bioprocessing (CBP). CBP uses a single reactor to
produce cellulase enzymes, hydrolyze biomass, and ferment ethanol. It doesn't require expensive
inputs and is inexpensive and readily available. The microorganisms that are employed are
Paecilomyces species, Fusarium oxysporum, Neurospora crassa, and Clostridium thermocellum.
However, because to its lengthy fermentation times and low ethanol production coefficient, the
CBF approach is inefficient.

Current strategies concentrate on creating recombination yeast that is capable of breaking down
any type of sugar in order to increase ethanol output and lower operating expenses. This issue is
addressed by two methods: genetic engineering microbes to ferment both pentose and hexose
sugars, and altering the genetic composition of microorganisms and their metabolic pathways. At
the moment, recombinant E. coli KO11 and P. stipitis BCC15191 are examples of genetically
engineered microorganisms. Depending on variables including enzyme kinetics, the kind of

20
lignocellulosic hydrolysate, and cost-effective inputs, fermentation operations can be fed in
batches, batch-wise, or continuously.

Feedstock crop
Compaction,
Production
Transportation &
storage

Pretreatment
processing

Production of
Detoxification &
enzyme in
neutralization bioreactor

Solid & liquid Enzymatic

separation hydrolysis of cellulose

& hemicellulose

Fermentation of
hemicellulose Fermentation of
sugar cellulose

Product recovery

Residue recovery
Ethanol
for co-product

Fig.3 Flowchart of cellulosic ethanol production from lignocellulosic biomass

21
 Chapter Three
3. Materials and methods
The transformation of lignocellulosic biomass from food processing activities into bioethanol
involves a series of steps and the use of specific materials and methods.

3.1 Materials:
1. Lignocellulosic biomass: This includes various agricultural residues such as wheat straw, rice
straw, corn stover, sugarcane bagasse, and other crop residues obtained from food processing
activities.

2. Pretreatment chemicals: Various chemicals such as dilute acid, alkali, or enzymes are used
for pretreatment of the biomass to remove lignin and hemicellulose, thus facilitating the access
of enzymes to cellulose.

3. Enzymes: A combination of cellulase and hemicellulase enzymes is used to break down

cellulose and hemicellulose into fermentable sugars.

4. Microorganisms: Ethanol-producing microorganisms, such as Saccharomyces cerevisiae or

other yeast strains, are required for the fermentation process.

5. Fermentation media: Nutrient-rich media containing carbohydrates, nitrogen sources,

vitamins, and minerals are used to support the growth and ethanol production by the
microorganisms.

3.2 Methods:
1. Pretreatment: Lignocellulosic biomass is subjected to a pretreatment process, such as dilute
acid, alkali, or enzymatic pretreatment, to break down the lignin and hemicellulose, making
cellulose more accessible to enzymes.

2. Enzymatic hydrolysis: Pretreated biomass is hydrolyzed using cellulase and hemicellulase

enzymes, which convert cellulose and hemicellulose into glucose and other fermentable sugars.

3. Fermentation: The hydrolyzed sugars are then subjected to fermentation by adding the
selected ethanol-producing microorganisms. These microorganisms consume the sugars and
produce ethanol as a metabolic byproduct.

22
4. Distillation: The fermented broth is then subjected to distillation to separate and concentrate
the ethanol.

5. Dehydration: The concentrated ethanol obtained from distillation is further dehydrated to

increase its purity and remove any remaining water content.

6. Ethanol recovery: The dehydrated ethanol is finally recovered and purified through various
techniques, such as molecular sieves or distillation, to obtain high-purity bioethanol.

Overall, the transformation of lignocellulosic biomass from food processing activities into
bioethanol requires specific materials, including lignocellulosic biomass, pretreatment chemicals,
enzymes, microorganisms, and fermentation media. The process involves several steps, including
pretreatment, enzymatic hydrolysis, fermentation, distillation, dehydration, and ethanol recovery.

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Conclusion
Growing in prominence as a readily available and affordable source of fermentable sugars for the
generation of liquid fuel is lignocellulosic biomass. A costly process in the synthesis of
bioethanol from this kind of biomass is the enzymatic treatment after pretreatment. A lot of
research has been done to find ways to reduce the amount of sugar that is broken down during
the pretreatment process, increase the recovery of fermentable carbohydrates, use less energy and
chemicals, create valuable byproducts, and lower the cost of the bioethanol process. Pretreatment
methods are generally categorized into four main groups: chemical, biological, physical-
chemical, and physical. While each approach has some benefits, not all biomass types can be
handled by a single approach. Selecting the optimal pretreatment method or combinations for a
given biomass feedstock would be greatly aided by having a basic understanding of the various
pretreatment technologies, the various compositions of biomass feedstock, and the relationship
between the composition of biomass feedstock and pretreatment methods. However, recent
developments in the fields of functional genomics, metagenomics, genetic engineering, and
metabolic engineering suggest that the development of artificially engineered plants with high
cellulose content and hydrolase production capabilities will be crucial to the commercial
production of bioethanol from food processing activities in the future.

24
Summary
The transformation of lignocellulosic biomass from food processing activities into bioethanol
involves the conversion of non-edible agricultural residues, such as crop stalks, rice husks, and
wheat straw, into a valuable source of renewable energy.

Three main components make up lignocellulosic biomass: cellulose, hemicellulose, and lignin.
Before these ingredients can be fermented into bioethanol, they must first be divided into smaller
molecules. A number of pretreatment techniques are used to separate the components and
increase their accessibility to enzymes, including enzymatic hydrolysis, acid hydrolysis, and
steam explosion.

Enzymes like cellulases and hemicellulases can be used to transform cellulose and hemicellulose
into fermentable sugars following pretreatment. These enzymes decompose complex
polysaccharides into monosaccharides like glucose, xylose, and arabinose. Specific strains of
microbes, usually yeast or bacteria, can then ferment these monosaccharides further to produce
bioethanol. But lignin presents a problem since it is very difficult to break down and can impede
fermentation and enzymatic hydrolysis. To get around this problem, a number of tactics are
being investigated and developed, including the removal and modification of lignin.

The process of turning lignocellulosic biomass into bioethanol has several advantages. It
decreases the demand for fossil fuels, promotes the use of sustainable and circular economic
methods, and aids in the reduction of greenhouse gas emissions. Using agricultural wastes as a
feedstock also makes use of waste materials that would otherwise be thrown away and lessens
competition with food production.

Despite encouraging potential, large-scale bioethanol production from lignocellulosic biomass

still faces technological and financial obstacles. To maximize conversion efficiency and make
the manufacture of bioethanol from agricultural wastes economically feasible, research and
innovation are being conducted in the areas of lignin valorization, fermentation, enzymatic
hydrolysis, and pretreatment techniques.

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Refrence
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07578-5_8, © Springer International Publishing Switzerland 2014

Su, T. et al. (2020) ‘Lignocellulosic biomass for bioethanol: Recent advances, technology trends,
and barriers to industrial development’, Current Opinion in Green and Sustainable Chemistry,
24, pp. 56–60. Available at: https://doi.org/10.1016/j.cogsc.2020.04.005.

Tayyab, M. et al. (2018) ‘Bioethanol production from lignocellulosic biomass by environment-

friendly pretreatment methods: A review’, Applied Ecology and Environmental Research, 16(1),
pp. 225–249. Available at: https://doi.org/10.15666/aeer/1601_225249.

Devendra Prasad Maurya • Ankit Singla • Sangeeta Negi et al. (2015), An overview of key
pretreatment processes for biological conversion of lignocellulosic biomass to bioethanol.

S. Haghighi Mood et al. / Renewable and Sustainable Energy Reviews 27 (2013) 77–93.
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